U.S. patent application number 15/444335 was filed with the patent office on 2018-03-22 for cell line stably expressing nav1.5 and method of screening therapeutic agent using the cell line.
This patent application is currently assigned to THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION. The applicant listed for this patent is THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION. Invention is credited to Jeong Hee CHOI, Jin-Sung CHOI.
Application Number | 20180080924 15/444335 |
Document ID | / |
Family ID | 61617508 |
Filed Date | 2018-03-22 |
United States Patent
Application |
20180080924 |
Kind Code |
A1 |
CHOI; Jin-Sung ; et
al. |
March 22, 2018 |
CELL LINE STABLY EXPRESSING Nav1.5 AND METHOD OF SCREENING
THERAPEUTIC AGENT USING THE CELL LINE
Abstract
Provided are a cell line stably expressing voltage-gated sodium
channel (Nav1.5), wherein a SCN5A gene (NCBI accession no.
NM_198056) encoding Nav1.5 and a puromycin selection marker are
inserted into a chromosome of a host cell, and a method of
measuring cardiotoxicity of a test substance comprising: (1)
contacting a test substance to the cell line of claim 1; and (2)
measuring magnitude of voltage-gated sodium channel (Nav1.5) in the
cell line in contact with the test substance.
Inventors: |
CHOI; Jin-Sung; (Seoul,
KR) ; CHOI; Jeong Hee; (Cheongju-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION
FOUNDATION |
Seoul |
|
KR |
|
|
Assignee: |
THE CATHOLIC UNIVERSITY OF KOREA
INDUSTRY-ACADEMIC COOPERATION FOUNDATION
Seoul
KR
|
Family ID: |
61617508 |
Appl. No.: |
15/444335 |
Filed: |
February 28, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/705 20130101;
G01N 33/6872 20130101; G01N 2500/10 20130101; G01N 2500/04
20130101; G01N 33/5014 20130101; C07K 14/705 20130101; G01N
33/48728 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/487 20060101 G01N033/487; C07K 14/705 20060101
C07K014/705; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2016 |
KR |
10-2016-0121472 |
Claims
1. A cell line stably expressing voltage-gated sodium channel
(Nav1.5), wherein a SCN5A gene (NCBI accession no. NM_198056)
encoding Nav1.5 and a puromycin selection marker are inserted into
a chromosome of a host cell.
2. The cell line of claim 1, wherein the cell line has accession
number KCTC 13095BP.
3. The cell line of claim 1, wherein the cell line has current
magnitude in a range from about 5.5 nA to about 19.6 nA.
4. The cell line of claim 3, wherein the cell line has average
current quantity of about 8.8.+-.0.8 nA.
5. The cell line of claim 1, wherein the host cell is a human
embryonic kidney 293 (HEK293) cell.
6. A method of measuring cardiotoxicity of a test substance, the
method comprising: (1) contacting a test substance to the cell line
of claim 1; and (2) measuring magnitude of voltage-gated sodium
channel (Nav1.5) in the cell line in contact with the test
substance.
7. A method of screening an inhibitor for cardiotoxicity, the
method comprising: (1) contacting a test substance to the cell line
of claim 1; (2) measuring magnitude of voltage-gated sodium channel
(Nav1.5) in the cell line in contact with the test substance; and
(3) selecting the test substance having increased magnitude of
Nav1.5 current as compared to magnitude of Nav1.5 current in a
control sample.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Korean Patent
Application No. 10-2016-0121472, filed on Sep. 22, 2016, in the
Korean Intellectual Property Office, the disclosure of which is
incorporated herein in its entirety by reference.
BACKGROUND
1. Field
[0002] The present disclosure relates to a cell line that stably
expresses human voltage-gated sodium channel (Nav1.5) (description:
Homo sapiens sodium channel, voltage-gated, type V, alpha subunit
(SCN5A), transcript variant 1), which is a expressed in cardiac
muscle of humans, and a method of screening a therapeutic agent
using the cell line.
2. Description of the Related Art
[0003] Many drugs that cause cardiovascular diseases have been
found to be a major part among drugs that have been withdrawn over
the last 15 years, and accordingly, the need for safety
pharmacology tests for cardiovascular diseases has been raised. The
ultimate competitiveness of most therapeutic agents discovered in
the development of new drugs depends on the `safety` for humans.
However, currently, many therapeutic agents are found to have side
effects such as ventricular repolarization and arrhythmia. In
particular, cardiac toxicity which is acute toxicity is a very
important safety factor, and thus the safety thereof in humans is
predicted through animal experiments in the non-clinical state of
the development of new drugs. However, ethical problems of animal
experiments have to be considered in the case of the toxicity test
using animals, and results obtained from the toxicity test using
animals may be different from the safety for humans. Recently,
rather than experimental animals, cell lines expressing main
proteins in humans are used to measure drug safety.
[0004] In the development of a therapeutic agent, suppression of
Nav1.5 current by a new drug may cause side effects such as acute
heart attack, and thus, the degree of suppression of the current
may be measured. However, if the magnitude of the current is small,
the ratio of signal-to-noise (S/N) is lowered due to experimental
errors such as leak current or noise, and accordingly, the
reliability of the measured value is also lowered. Therefore, as
long as the magnitude of the current does not exceed a maximum
effective range that the experimental machine can measure, the
larger magnitude the current produced by a cell line has, the
higher the S/N ratio is, so that the more reliable experimental
data can be obtained. However, a cell line having a sufficiently
large current magnitude of Na.sub.v1.5 has not yet been
developed.
DOCUMENT IN RELATED ARTS
Patent Document
[0005] WO 2010/071983 (published on Jul. 1, 2010)
SUMMARY
[0006] Provided is a cell line stably expressing human
voltage-gated sodium channel (Nav1.5).
[0007] Provided is a method of measuring cardiotoxicity by
measuring magnitude of Na.sub.v1.5 current in the cell line.
[0008] Provided is a method of screening an inhibitor for
cardiotoxicity by measuring magnitude of Nav1.5 current in the cell
line.
[0009] Additional aspects will be set forth in part in the
description which follows and, in part, will be apparent from the
description, or may be learned by practice of the presented
embodiments.
[0010] In an embodiment, there is provided a cell line stably
expressing human voltage-gated sodium channel (Nav1.5), the cell
line being produced in a way that a SCN5A gene (NCBI accession no.
NM_198056) encoding Nav1.5 and a puromycin selection marker are
inserted into a chromosome of a host cell.
[0011] In an embodiment, there is provided a method of measuring
cardiotoxicity of a test substance, the method including: (1)
contacting a test substance to a cell line; and (2) measuring
magnitude of Nav1.5 current in the cell line in contact with the
test substance.
[0012] In an embodiment, there is provided a method of screening an
inhibitor for cardiotoxicity, the method including: (1) contacting
a test substance to a cell line; (2) measuring magnitude of Nav1.5
current in the cell line in contact with the test substance; and
(3) selecting the test substance having increased magnitude of
Nav1.5 current as compared to magnitude of Nav1.5 current in a
control sample.
BRIEF DESCRIPTION OF THE DRAWING
[0013] These and/or other aspects will become apparent and more
readily appreciated from the following description of the example
embodiments, taken in conjunction with the accompanying drawing in
which:
[0014] FIG. 1 shows graphs each showing hNav1.5 current and
current-voltage curve;
[0015] FIG. 2 is a graph showing voltage-dependent activation curve
and steady state voltage-dependent inactivation curve; and
[0016] FIG. 3 shows graphs each showing inhibitory results of
hNav1.5 current by lidocaine at a holding potential (HP) of -120 mV
and -90 mV.
DETAILED DESCRIPTION
[0017] Reference will now be made in detail to embodiments,
examples of which are illustrated in the accompanying drawings,
wherein like reference numerals refer to like elements throughout.
In this regard, the present embodiments may have different forms
and should not be construed as being limited to the descriptions
set forth herein. Accordingly, the embodiments are merely described
below, by referring to the figures, to explain aspects.
[0018] An embodiment of the present inventive concept provides a
cell line stably expressing human voltage-gated sodium channel
(Nav1.5), the cell line being produced in a way that a SCN5A gene
(NCBI accession no. NM_198056) encoding Nav1.5 and a puromycin
selection marker are inserted into a chromosome of a host cell.
[0019] In an embodiment, the cell line stably expressing Nav1.5 may
have accession number KCTC 13095BP, but embodiments are not limited
thereto.
[0020] In an embodiment, the cell line stably expressing Nav1.5 may
have current magnitude in a range from about 5.5 nA to about 19.6
nA, and may have average current quantity of about 8.8.+-.0.8 nA.
However, embodiments are not limited thereto.
[0021] In an embodiment, the host cell may be a human embryonic
kidney 293 (HEK293) cell, but embodiments are not limited
thereto.
[0022] The term "SCN5A gene encoding Nav1.5" as used herein may
refer to transcript variant 1 (Q1077; NM_198056) among a total of 6
SCN5A transcript variants, and may be the longest variant that is
translated into 1,406 amino acids and cases no mutations (mRNA
Refseq: NM_198056.1, Protein Refseq: NP_932173).
[0023] An embodiment of the present inventive concept provides a
measuring cardiotoxicity of a test substance, the method including:
(1) contacting a test substance to a cell line; and (2) measuring
magnitude of Nav1.5 current in the cell line in contact with the
test substance.
[0024] An embodiment of the present inventive concept provides a
method of screening an inhibitor for cardiotoxicity, the method
including: (1) contacting a test substance to a cell line; (2)
measuring magnitude of Nav1.5 current in the cell line in contact
with the test substance; and (3) selecting the test substance
having increased magnitude of Nav1.5 current as compared to
magnitude of Nav1.5 current in a control sample.
[0025] The term "test substance" as used herein in connection with
the screening method may refer to an unknown candidate substance
used in the screening method to determine whether the unknown
candidate substance influences magnitude of Nav1.5 current. The
test substance may include a compound, a nucleotide, an
antisense-RNA, a small interference RNA (siRNA), and a natural
extract, but embodiments are not limited thereto.
[0026] Hereinafter, to promote understanding of one or more
embodiments, the inventive concept will be described more fully
with reference to Examples below. However, Examples shown and
described herein are illustrative examples of the inventive concept
and are not intended to otherwise limit the scope of the inventive
concept in any way. These Examples are provided so that this
disclosure will be thorough and complete, and will fully convey the
concept of the inventive concept to those skilled in the art.
<Example> Manufacture of Cell Line Stably Expressing
Nav1.5
[0027] 1. Manufacture of Stable Cell Line
[0028] To manufacture HEK293 cell line that stably expresses human
Nav1.5 (hNav1.5), a plasmid including a SCN5A gene (transcript
variant 1; Q1077; NM_198056) that encodes hNav1.5 was purchased
from OriGene Technologies, Inc., and then, was subjected to PCR
amplification. The amplified genes were sub-cloned into
pSF-CMV-puro vector (available from Sigma), and then, transfected
with HEK293 cells. Puromycin was applied thereto for 3 weeks in
terms of selection and induction of SCN5A stable cell line derived
from a single cell. Among cell lines that stably express Nav1.5
current, electrophysiological techniques were used to screen and
establish a cell line of which biophysical characteristics of
Nav1.5 current are the same as those of hNav1.5 current that has
been temporarily expressed.
[0029] 2. Electrophysiology
[0030] For electrophysiological analysis of hNav1.5 current,
current was measured by using Axopatch 200B (manufactured by
Molecular Devices Company) and Patchliner Octo (manufactured by
Nanion Company) for the manual patch-clamp recording and the
automated patch-clamp recording, respectively. Here, the
composition (mM) of the extracellular fluid used herein included
140 NaCl, 3 KCl, 1 CaCl.sub.2, 1 MgCl.sub.2, and 10 HEPES (pH 7.3,
NaOH), and the composition (mM) of the intracellular fluid included
140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES (pH 7.3, CsOH).
[0031] (1) Manual Patch-Clamp Recording Method
[0032] A glass electrode having a resistance value in a range from
about 0.7 M.OMEGA. to about 1.5 M.OMEGA. in the case of addition of
the intracellular fluid using a horizontal microelectrode puller
was prepared. Then, the glass electrode was adhered to a cell
through whole-cell configuration, thereby recording hNav1.5
current. hNav1.5 current data were stored in a computer through
Digidata 1440A interface. A pClamp 10.5 software was used to record
and analyze data. To minimize the distortion of the recorded
hNav1.5 current, only cells having a series resistance of about 1.5
M.OMEGA. or less during the whole-cell configuration were used. If
the current was greater than about 5 nA, 80% of the series
resistance was compensated. The recording of current was started 5
minutes after the whole-cell configuration was used, so as to
obtain stable current. Here, liquid junction potential was not
corrected. To verify pharmacological effects of lidocaine,
extracellular fluid or extracellular fluid containing lidocaine at
various concentrations was continuously perfused and recorded using
a perfusion pencil.
[0033] (2) Automated Patch-Clamp Recording Method
[0034] NPC.RTM.-16 chips having low resistance (about 1.5 M.OMEGA.
to about 2.0) were placed on NPC-16 Patchliner Octo platform
(Nanion Technologies GmbH) using two EPC 10 quadro patch-clamp
amplifiers to measure and record hNav1.5 current from the cells.
Here, current was automatically recorded by using a PatchControlHT
software, and the recorded current was analyzed by using a
Patchmaster software.
[0035] 3. Biophysical Characteristics hNav1.5 Current
[0036] First, to accurately identify biophysical characteristics of
hNav1.5 current, the manual patch-clamp recording method was used.
To measure the maximum current that cells can generate, a holding
potential was set to about -120 mV, and the cells were depolarized
at an interval of about 5 mV from about -90 mV to about +40 mV for
40 ms every 10 seconds. Then, current generated in the cells
undergone the stimulation was recorded (see FIG. 1). In all the
cells being measured (n=12), the current was generated in a range
from a minimum of about 5.5 nA to a maximum of about 19.6 nA, and
average current quantity of the cells was about 8.8.+-.0.8 nA. To
eliminate the difference in the current quantity depending on the
cell size, the measured current quantity was divided by unit area
of the cells (i.e., capacitance of the cells), to thereby measure
current density. The current density measured herein was about 319
pA/pF at a minimum and about 921 pA/pF at a maximum, and the
average current density was about 556.+-.45 pA/pF. To determine the
current quantity of the cells at each voltage, a current-voltage
curve (see right of FIG. 1) was plotted, and accordingly, it was
found that maximum current appeared at a voltage of about -30 mV
and reversal potential appeared at a voltage of about +57.7 mV, in
a similar manner as in the equilibrium potential of sodium
current.
[0037] The voltage-dependant activation curve (see left of FIG. 1)
was optimized according to the Boltzmann distribution below, and
more particularly, reversal potential obtained from the
current-voltage curve was used to calculate conductance (G) which
was to be divided by a maximum value:
G = G max 1 + e V 50 - V k ##EQU00001##
[0038] Here, V.sub.50 indicates voltage that is 50% of a maximum
value, and V.sub.50 of Nav1.5 current of about -47.15.+-.0.59
mV.
[0039] The steady state voltage-dependent inactivation curve (see
right axis of FIG. 2) was optimized according to the Boltzmann
distribution below, and more particularly, current generated at -10
mV for 40 ms after applying stimulation thereto with voltage
increased by 10 mV from about -180 mV to about -10 mV for 500 ms
every 10 seconds at holding potential of about -120 mV was divided
by a maximum value:
I I 0 = 1 1 + e V 50 - V k ##EQU00002##
[0040] Here, V.sub.50 voltage that is 50% of a maximum value, and
V.sub.50 of hNav1.5 current in the steady state voltage-dependent
inactivation curve was about -92.80.+-.4.53 mV.
[0041] To measure inhibitory effects of lidocaine on hNav1.5
current, the cells were depolarized at an interval of -20 mV for 40
ms every 5 seconds at holding potential of about -120 mV where
hNav1.5 current was not inactivated (upper left of FIG. 3) and at
holding potential of about -90 mV which is a similar level with
stable potential of the cardiac muscle (see right upper of FIG. 3),
thereby recording hNav1.5 current. Here, the flow of lidocaine at
various concentrations was added to the cells, to thereby measure
the degree to which the peak of the current was inhibited. The
magnitude of the inhibited current was optimized according to the
Hill equation, and then, the concentration of lidocaine at which
the current was inhibited by 50% was calculated. Consequently, it
was found that the concentration at which inhibition of hNav1.5
current by lidocaine appeared was about 775.64.+-.37.10 .mu.M at
-120 mV (see left bottom of FIG. 3) and about 18.42.+-.2.60 .mu.M
at -90 mV (see right bottom of FIG. 3).
[0042] [Accession Number]
[0043] Name of depository institution: Korean Research Instituted
of Bioscience and Biotechnology
[0044] Accession number: KCTC 13095BP (Receipt of microorganism
deposit is appended to this disclosure)
[0045] Accession date: Sep. 1, 2016
[0046] According to one or more embodiments of the present
inventive concept, a cell line stably expressing Nav1.5 and a
method of screening a therapeutic agent using the cell line are
disclosed. The inventors of the present inventive concept developed
a new cell line in a way that hNav1.5, which is a sodium channel
mainly expressed in human cardiomyocytes, was stably expressed in a
human embryonic kidney 293 (HEK293) cell. Here, the new cell line
was developed by using automated electrophysiological devices as
well as traditional electrophysiological techniques, to thereby
measure toxicity of drugs. Therefore, the cell line of the present
inventive concept can be effectively utilized for screening
cardiotoxicity of pharmaceutical products.
[0047] It should be understood that the example embodiments
described herein should be considered in a descriptive sense only
and not for purposes of limitation. Descriptions of features or
aspects within each example embodiment should typically be
considered as available for other similar features or aspects in
other embodiments.
[0048] While one or more example embodiments have been described
with reference to the figures, it will be understood by those of
ordinary skill in the art that various changes in form and details
may be made therein without departing from the spirit and scope as
defined by the following claims.
* * * * *